PROJECT SUMMARY Lyme disease is the most commonly reported arthropod-borne infection in the US with recent CDC estimates eclipsing 300,000 new cases in 2013. In addition to growing in frequency, the infections have a complex and increasingly severe course. Beginning with mild flu-like symptoms and frequently a signature bull?s-eye rash, erythema migrans, Lyme disease can progress to severe articular, neurological and cardiac symptoms, most of which are preventable with early antibiotic therapy. Leading investigators have identified two major shortcomings to the current serology-based methods for the definitive diagnosis of Early Localized Lyme disease. First, the clinical sensitivity in the first four weeks is poor, under 50% at the time of symptom onset, so many patients remain undiagnosed or unconfirmed until the disease has had time to progress. Second, serum antibody levels remain elevated long after the infection has been resolved making the monitoring of therapeutic success or diagnosis of re-infection virtually impossible. We will address these shortcomings by changing the sample matrix that is used for diagnosis. Early in an infection pathogen-specific B lymphocytes are activated to form plasmablasts or antibody-secreting cells (ASCs) that enter and transiently circulate in blood. These circulating ASCs are present in the blood only during active infection and their numbers decline rapidly when the infection is resolved. By harvesting ASCs, and culturing them in a virgin cell culture medium, the ASCs create a novel matrix, MENSA (medium enriched for newly synthesized antibody). MENSA is an ideal analytical medium; it contains easily measurable quantities of newly synthesized, pathogen-specific antibodies and it has no antibodies from prior infections. ASCs emerge into the blood prior to seroconversion, so measurement of anti-B. burgdorferi antibodies in MENSA will facilitate earlier diagnosis of Lyme disease. Unlike serum antibodies, circulating ASCs disappear when the infection resolves, so levels of anti-B. burgdorferi antibodies in MENSA can be used to track the success of antibiotic therapy. MicroB-plex and its clinical collaborators have reduced to practice special skills needed to demonstrate these major improvements in Lyme diagnostics using human subjects. These include definitive commercial ELISAs and custom-made high sensitivity anti-B. burgdorferi antibody assays validated using a CDC sample panel, recruitment of newly infected subjects in endemic areas, shipment of whole blood from clinical sites in Maine and Maryland to Microbplex? laboratory in Atlanta, Georgia for processing and analysis, and preliminary data showing that antibody levels in MENSA will track therapeutic success. The two Specific Aims focus directly on early diagnosis (Aim 1) and tracking therapy (Aim 2) by following subjects with early Lyme disease through diagnosis and therapy over a period of months. Success will create important new opportunities for diagnosis of Lyme disease and can be extended to other infectious diseases.